本文選題：對(duì)乙酰氨基酚　切入點(diǎn)：藥物性肝損傷　出處：《安徽醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：背景與目的藥物性肝損傷(drug induced liver injury,DILI)嚴(yán)重威脅患者用藥后的生命安全。對(duì)乙酰氨基酚(acetaminophen,APAP)是臨床常用的消炎鎮(zhèn)痛藥物,在過量使用時(shí)可引起急性肝衰竭甚至死亡。APAP造成肝損傷的機(jī)制可能有炎癥反應(yīng)、氧化應(yīng)激、線粒體自噬、內(nèi)質(zhì)網(wǎng)應(yīng)激等機(jī)制。過氧化物酶體增殖物激活受體γ(peroxisome proliferator activated receptors,PPARγ)具有調(diào)節(jié)脂質(zhì)代謝、調(diào)節(jié)免疫、抗腫瘤等生理功能,對(duì)多種因素引起的肝損傷具有保護(hù)作用。噻唑烷二酮類藥物(troglitazone,TZDs)是PPARγ的配體,可以與PPARγ結(jié)合并激活PPARγ信號(hào)通路。已有多篇文獻(xiàn)報(bào)道,PPARγ激動(dòng)劑羅格列酮(Rosiglitazone,RSG)具有抗炎及抗氧化應(yīng)激的作用,但RSG是否可以減輕APAP急性肝損傷時(shí)的炎癥及氧化應(yīng)激反應(yīng)尚不清楚。本研究擬在建立APAP誘導(dǎo)急性肝損傷動(dòng)物模型的基礎(chǔ)上,觀察RSG對(duì)APAP誘導(dǎo)急性肝損傷的保護(hù)作用,并從炎癥反應(yīng)、氧化應(yīng)激機(jī)制方面初步探討RSG對(duì)APAP急性肝損傷保護(hù)作用的機(jī)理。方法1.為觀察RSG對(duì)APAP急性肝損傷的保護(hù)作用,隨機(jī)將健康雄性CD-1小鼠分成10組,每組6只,共60只。正常對(duì)照組:正常飼養(yǎng),在APAP組給藥的同時(shí)用生理鹽水腹腔注射;單純RSG組:在APAP組給藥前48h、24h及1h分別予以RSG(20mg/kg)灌胃,在APAP組給藥的同時(shí)給予等容積的生理鹽水腹腔內(nèi)注射;APAP組(4組,分別為APAP 0h組、APAP 1h組、APAP 4h組及APAP 24h組):單次給予APAP(300mg/Kg)腹腔注射;RSG+APAP組(4組,即RSG+APAP 0h組、RSG+APA P1h組、RSG+APAP 4h組及RSG+APAP 24h組):在腹腔注射APAP前48 h、24h和1h予以RSG(20mg/kg)各灌胃一次。APAP組及RSG+APAP組分別在APAP給藥后0h、1h、4h、24h剖殺小鼠。所有小鼠在APAP給藥前禁食12 h。收集血清檢測(cè)ALT、AST、CRP和TBIL水平;部分肝臟組織行HE病理檢查及凋亡檢測(cè)。2.為研究RSG對(duì)APAP誘導(dǎo)的急性肝損傷小鼠生存率的影響,隨機(jī)將小鼠分為兩組,每組10只。APAP組及RSG+APAP組處理方法同方法1。觀察兩組小鼠在1周內(nèi)每天的生存?zhèn)�數(shù),繪制生存曲線。3.為觀察RSG對(duì)APAPⅠ相代謝酶CYP2E1的影響,隨機(jī)將小鼠分成4組,每組6只,即正常對(duì)照組、RSG組、APAP組、RSG+APAP組,處理方法同方法1,在APAP處理后0.5h剖殺各組小鼠,取肝臟組織用Western blotting法檢測(cè)CYP2E1的表達(dá)。4.為觀察RSG對(duì)APAP急性肝損傷的肝臟組織GSH代謝酶活性的影響,隨機(jī)將小鼠分成4組,每組6只,共24只,即正常對(duì)照組、RSG組、APAP組、RSG+APAP組,處理方法同方法1,在APAP給藥后1h剖殺各組小鼠,取新鮮肝組織檢測(cè)谷胱甘肽-S-轉(zhuǎn)移酶(glutathione S-transferase,GST)、谷胱甘肽還原酶(glutathione reductase,GSH-Rd)和谷胱甘肽過氧化物酶(Glutathione peroxidase,GSH-Px)的酶活性。結(jié)果1.RSG對(duì)APAP急性肝損傷的保護(hù)作用從APAP給藥后1h開始,血清ALT、AST水平即進(jìn)行性升高。血清TBIL水平從APAP處理后4h開始進(jìn)行性升高。RSG預(yù)處理后,血清ALT、AST及TBIL水平較同時(shí)段單純APAP組有不同程度的降低。HE染色結(jié)果顯示,在APAP 1h組,肝臟開始出現(xiàn)淤血水腫;APAP4h組有明顯的肝細(xì)胞點(diǎn)狀壞死、橋接壞死及亞大塊壞死;在APAP 24h組出現(xiàn)明顯的大塊狀壞死。用TUNEL技術(shù)檢查發(fā)現(xiàn),在APAP 1h組,可見有少許肝胞凋亡;APAP 4h組,其凋亡陽性細(xì)胞數(shù)則明顯增加,在24h達(dá)到最高峰。RSG預(yù)處理的APAP組,肝細(xì)胞的變性、壞死程度及壞死面積均較同時(shí)段單純APAP組輕,且凋亡陽性細(xì)胞數(shù)也明顯低于同時(shí)段單純APAP組。單純APAP組小鼠7日生存率為50%,明顯低于RSG預(yù)處理組(80%)。3.RSG對(duì)APAPⅠ相代謝酶CYP2E1的代謝的影響APAP急性肝損傷時(shí)CYP2E1的表達(dá)并未發(fā)生明顯變化,RSG也不影響急性APAP急性肝損傷時(shí)的CYP2E1的表達(dá)。4.RSG對(duì)APAP急性肝損傷炎癥反應(yīng)的影響4.1 RSG對(duì)APAP急性肝損傷模型的血清C反應(yīng)蛋白(C-reactive protein,CRP)的影響與對(duì)照組相比,APAP組在4h后血清CRP表達(dá)開始輕度升高,在24h達(dá)高峰;用RSG預(yù)處理可以降低APAP造成的CRP升高水平。4.2 RSG對(duì)APAP急性肝損傷時(shí)有絲分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信號(hào)通路的影響用Western-blotting技術(shù),對(duì)MAPK亞家族成員JNK、ERK1/2、P38MAPK及AKT的磷酸化水平進(jìn)行觀察。結(jié)果提示,APAP急性肝損傷過程中,上述4種MAPK家族分子均被磷酸化激活。而使用RSG預(yù)處理,可以明顯下調(diào)APAP誘導(dǎo)的上述分子的磷酸化水平。4.3 RSG對(duì)APAP急性肝損傷時(shí)NF-κB的信號(hào)通路的影響用Western blotting技術(shù)檢測(cè)發(fā)現(xiàn),在APAP給藥后1h,細(xì)胞核內(nèi)的P65和P50表達(dá)增加,并且進(jìn)行性升高至24h。表明NF-κB在APAP給藥后早期即被激活。使用RSG預(yù)處理,可以明顯下調(diào)NF-κB的核表達(dá)。4.4 RSG對(duì)APAP急性肝損傷時(shí)炎性相關(guān)因子表達(dá)的影響用RT-PCR的方法觀察RSG對(duì)APAP急性肝損傷時(shí)TNFα、KC、COX-2等炎性因子或趨化因子表達(dá)的影響。結(jié)果發(fā)現(xiàn),與對(duì)照組相比,APAP組在出現(xiàn)肝損傷時(shí),細(xì)胞因子TNFα、KC的m RNA水平明顯增加,而RSG預(yù)處理的APAP組,TNFα、KC的m RNA水平明顯低于同時(shí)段的APAP組。而各組COX-2的轉(zhuǎn)錄情況并沒有明顯差別。5.RSG對(duì)APAP誘導(dǎo)肝損傷氧化應(yīng)激通路的影響5.1 RSG可以減輕APAP導(dǎo)致的還原型谷胱甘肽(glutathione,GSH)耗竭2.RSG對(duì)APAP誘導(dǎo)的急性肝損傷小鼠生存率的影響在APAP1h組,其肝臟組織中的GSH處于耗竭狀態(tài);在APAP4h組,其肝組織中GSH含量較APAP1h組有所上升;在APAP 24h組,GSH幾乎恢復(fù)至對(duì)照組水平。在RSG+APAP 1h組,GSH水平明顯高于單純APAP組;而在RSG+APAP 4h及RSG+APAP 24h組,其肝臟GSH含量與正常對(duì)照組無明顯差別。5.2 RSG對(duì)APAP急性肝損傷的肝臟組織GSH代謝酶活性的影響APAP過量可導(dǎo)致GST、GSH-Rd、GSH-Px三種代謝酶活性降低,RSG可以改善由APAP造成的GSH-Rd的活性減低,對(duì)于GST及GSH-Px活降低也有一定的恢復(fù)作用,但無明顯的統(tǒng)計(jì)學(xué)意義。5.3 RSG對(duì)APAP急性肝損傷脂質(zhì)過氧化產(chǎn)物表達(dá)的影響將肝臟組織進(jìn)行勻漿后,測(cè)定丙二醛(malonaldehyde,MDA)含量。結(jié)果發(fā)現(xiàn),在APAP肝損傷早期(1h)即有MDA含量的明顯升高;而在4h后MDA含量及正常組無明顯的變化,而使用RSG預(yù)處理組各時(shí)段其MDA含量均未見明顯升高。5.4 RSG對(duì)APAP急性肝損傷肝臟組織3硝基酪氨酸(3NT)的影響采用Western blotting技術(shù)檢測(cè)提示,從APAP給藥后1h開始,3NT的表達(dá)呈進(jìn)行性增加。而RSG預(yù)處理可以明顯降低APAP急性肝損傷組織的3NT表達(dá)。5.5 RSG對(duì)APAP急性肝損傷肝臟組織NADPH氧化酶(NADPH xidase,NOX)表達(dá)的影響用Western blotting技術(shù)檢測(cè)發(fā)現(xiàn),從APAP給藥后1h開始,NOX-2及NOX-4的表達(dá)呈進(jìn)行性升高,并且與肝損傷的程度相一致。而RSG預(yù)處理可以明顯降低APAP給藥后NOX-2及NOX-4的表達(dá)。結(jié)論綜合以上實(shí)驗(yàn)結(jié)果,可以得出以下結(jié)論:1.RSG對(duì)APAP急性肝損傷具有保護(hù)作用;2.RSG可能通過抑制炎癥反應(yīng)而發(fā)揮其對(duì)APAP急性肝損傷的保護(hù)作用;3.RSG對(duì)APAP急性肝損傷的保護(hù)作用可能與抑制氧化應(yīng)激反應(yīng)有關(guān)。
[Abstract]:Background and purpose of drug-induced liver injury (drug induced liver injury, DILI) treatment in patients with serious threat to life safety. After acetaminophen (acetaminophen, APAP) is a commonly used anti-inflammatory analgesic drugs, the excessive use can cause the mechanism of acute liver failure and even death.APAP caused liver injury may have inflammation, oxidation stress, mitochondrial autophagy, endoplasmic reticulum stress. The mechanism of peroxisome proliferator activated receptor gamma (peroxisome proliferator activated receptors, PPAR y) could regulate lipid metabolism, immune regulation, anti-tumor and other physiological functions, has a protective effect on liver injury caused by many factors. Two thiazolidinediones (troglitazone, TZDs) is a ligand PPAR gamma, and PPAR gamma combined activation of PPAR signal pathway of gamma. There are many reports, PPAR agonist rosiglitazone (Rosiglitazone, RSG) has anti-inflammatory and The role of oxidative stress, but whether RSG can reduce oxidative stress and inflammation in acute liver injury of APAP is not clear. This study based on the establishment of animal model of acute liver injury induced by APAP on the protection effects of RSG on APAP induced acute liver injury, and the inflammatory reaction, oxidative stress mechanism to explore the mechanism of protective effect of RSG on APAP induced acute liver injury. Methods 1. in order to observe the protective effect of RSG on APAP induced acute liver injury, random healthy male CD-1 mice were divided into 10 groups, 6 rats in each group, a total of 60. Normal control group: normal diet, medication in group APAP and saline intraperitoneal injection; RSG group: before administration of 48h in group APAP, 24h and 1H respectively to RSG (20mg/kg) by intragastric administration, given saline intraperitoneal injection volume in the APAP group; APAP group (Group 4, APAP respectively in 0h group, APAP 1H group, APAP group and APA 4H P 24h緇，

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